Erik S. Hopmans

Human and mouse oligonucleotide-based array CGH

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using ‘off-the-shelf’ oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.

Hierarchy in somatic mutations arising during genomic evolution and progression of follicular lymphoma.

Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies.

Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression

Objective: This study aimed to identify the oncogenes at 20q involved in colorectal adenoma to carcinoma progression by measuring the effect of 20q gain on mRNA expression of genes in this amplicon. Methods: Segmentation of DNA copy number changes on 20q was performed by array CGH (comparative genomic hybridisation) in 34 non-progressed colorectal adenomas, 41 progressed adenomas (ie, adenomas that present a focus of cancer) and 33 adenocarcinomas. Moreover, a robust analysis of altered expression of genes in these segments was performed by microarray analysis in 37 adenomas and 31 adenocarcinomas. Protein expression was evaluated by immunohistochemistry on tissue microarrays. Results: The genes C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5, mapping at 20q, were significantly overexpressed in carcinomas compared with adenomas as a consequence of copy number gain of 20q. Conclusion: This approach revealed C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5 genes to be important in chromosomal instability-related adenoma to carcinoma progression. These genes therefore may serve as highly specific biomarkers for colorectal cancer with potential clinical applications.

Haplotyping germline and cancer genomes with high-throughput linked-read sequencing

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer

BackgroundGastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.ResultsBoth the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1-/-; Tp53-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.ConclusionsWe document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

A programmable method for massively parallel targeted sequencing

We have developed a targeted resequencing approach referred to as Oligonucleotide-Selective Sequencing. In this study, we report a series of significant improvements and novel applications of this method whereby the surface of a sequencing flow cell is modified in situ to capture specific genomic regions of interest from a sample and then sequenced. These improvements include a fully automated targeted sequencing platform through the use of a standard Illumina cBot fluidics station. Targeting optimization increased the yield of total on-target sequencing data 2-fold compared to the previous iteration, while simultaneously increasing the percentage of reads that could be mapped to the human genome. The described assays cover up to 1421 genes with a total coverage of 5.5 Megabases (Mb). We demonstrate a 10-fold abundance uniformity of greater than 90% in 1 log distance from the median and a targeting rate of up to 95%. We also sequenced continuous genomic loci up to 1.5 Mb while simultaneously genotyping SNPs and genes. Variants with low minor allele fraction were sensitively detected at levels of 5%. Finally, we determined the exact breakpoint sequence of cancer rearrangements. Overall, this approach has high performance for selective sequencing of genome targets, configuration flexibility and variant calling accuracy.

RVD: a command-line program for ultrasensitive rare single nucleotide variant detection using targeted next-generation DNA resequencing

BackgroundRare single nucleotide variants play an important role in genetic diversity and heterogeneity of specific human disease. For example, an individual clinical sample can harbor rare mutations at minor frequencies. Genetic diversity within an individual clinical sample is oftentimes reflected in rare mutations. Therefore, detecting rare variants prior to treatment may prove to be a useful predictor for therapeutic response. Current rare variant detection algorithms using next generation DNA sequencing are limited by inherent sequencing error rate and platform availability.FindingsHere we describe an optimized implementation of a rare variant detection algorithm called RVD for use in targeted gene resequencing. RVD is available both as a command-line program and for use in MATLAB and estimates context-specific error using a beta-binomial model to call variants with minor allele frequency (MAF) as low as 0.1%. We show that RVD accepts standard BAM formatted sequence files. We tested RVD analysis on multiple Illumina sequencing platforms, among the most widely used DNA sequencing platforms.ConclusionsRVD meets a growing need for highly sensitive and specific tools for variant detection. To demonstrate the usefulness of RVD, we carried out a thorough analysis of the software’s performance on synthetic and clinical virus samples sequenced on both an Illumina GAIIx and a MiSeq. We expect RVD can improve understanding the genetics and treatment of common viral diseases including influenza. RVD is available at the following URL:http://dna-discovery.stanford.edu/software/rvd/.

A genome-wide approach for detecting novel insertion-deletion variants of mid-range size

We present SWAN, a statistical framework for robust detection of genomic structural variants in next-generation sequencing data and an analysis of mid-range size insertion and deletions (<10 Kb) for whole genome analysis and DNA mixtures. To identify these mid-range size events, SWAN collectively uses information from read-pair, read-depth and one end mapped reads through statistical likelihoods based on Poisson field models. SWAN also uses soft-clip/split read remapping to supplement the likelihood analysis and determine variant boundaries. The accuracy of SWAN is demonstrated by in silico spike-ins and by identification of known variants in the NA12878 genome. We used SWAN to identify a series of novel set of mid-range insertion/deletion detection that were confirmed by targeted deep re-sequencing. An R package implementation of SWAN is open source and freely available.

Emergence of Hemagglutinin Mutations During the Course of Influenza Infection

Influenza remains a significant cause of disease mortality. The ongoing threat of influenza infection is partly attributable to the emergence of new mutations in the influenza genome. Among the influenza viral gene products, the hemagglutinin (HA) glycoprotein plays a critical role in influenza pathogenesis, is the target for vaccines and accumulates new mutations that may alter the efficacy of immunization. To study the emergence of HA mutations during the course of infection, we employed a deep-targeted sequencing method. We used samples from 17 patients with active H1N1 or H3N2 influenza infections. These patients were not treated with antivirals. In addition, we had samples from five patients who were analyzed longitudinally. Thus, we determined the quantitative changes in the fractional representation of HA mutations during the course of infection. Across individuals in the study, a series of novel HA mutations directly altered the HA coding sequence were identified. Serial viral sampling revealed HA mutations that either were stable, expanded or were reduced in representation during the course of the infection. Overall, we demonstrated the emergence of unique mutations specific to an infected individual and temporal genetic variation during infection.

Abstract 4882: Megabase-scale phased haplotypes of genetic aberrations from whole cancer genome sequencing of primary colorectal tumors

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Cancer genomes contain multiple types of genetic aberrations that include mutations, deletions, copy number variants and chromosomal rearrangements. Despite advances in next generation sequencing, it remains a major challenge to delineate many of these somatic genomic alterations because of intrinsic complexity of cancer genomes. Haplotyping involves the assignment of genetic variants such as mutations and structural variants to specific segment of homologous chromosomes. Experimentally determined phasing of cancer genomes offers an opportunity to resolve complex genomic structures such as somatic rearrangements, aneuploidy composition and ongoing evolutionary changes. However, contiguous phasing of cancer genomes on a megabase (Mb) scale remains difficult to achieve with sequencing-based approaches. In this proof-of-concept study, we experimentally determined Mb-scale haplotypes of primary tumor samples via whole genome sequencing. To generate haplotypes, we employed an automated instrument that partitions long DNA fragments into hundreds of thousands of reactions, each of which incorporates a unique, nonrandom barcode into indexed sequencing libraries. Given need to amplify from sparse numbers of molecules and the high efficiency of the automated sequencing library construction process, the DNA requirements for each sample are less than 5 ng. We sequenced the genomes of primary colorectal cancer samples and their matched normal diploid DNA with an Illumina sequencer. We used the single nucleotide variants to generate Mb-scale haplotype blocks (N50 of 1.2 Mb) with phased haplotype block size of up to 11.3 Mb. We were able to delineate cancer genome haplotypes that cover allelic imbalances, copy number variations such as deletions and other genomic instability events. Structural variants were identified in the context of their position in specific chromosome homologues. Thus, we improved the characterization of somatic genetic aberrations using contiguity mapping and cancer genome haplotypes in the context of whole cancer genome sequencing. Overall, we demonstrated the feasibility and potential utility of conducting contiguous phased haplotypes in whole cancer genome sequencing from primary tumor samples. Citation Format: Billy Lau, John M. Bell, Michael Schnall-Levin, Mirna Jarosz, Erik Hopmans, Christina M. Wood, Grace X. Zheng, Kristina Giorda, Hanlee P. Ji. Megabase-scale phased haplotypes of genetic aberrations from whole cancer genome sequencing of primary colorectal tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4882. doi:10.1158/1538-7445.AM2015-4882